Compounds for the treatment of pathologies associated with aging and degenerative disorders

ABSTRACT

The present invention relates to methods of inhibiting one or more signs of aging and/or degenerative disorder in a subject in need of such treatment, which comprise administering, to the subject, an effective amount of one or more of the compounds as set forth herein. “Inhibiting a sign of aging or degenerative disorder” means reducing the risk of occurrence, delaying the onset, slowing the progression, and/or reducing the severity and/or manifestation, of a sign of aging or degenerative disorder, and includes, but is not limited to, preventing the occurrence, development or progression of a sign of aging or degenerative disorder.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of International Application No.PCT/US2009/050869, filed Jul. 16, 2009, which claims priority to U.S.Provisional Patent Application Ser. No. 61/081,678, filed Jul. 17, 2008,both of which are hereby incorporated by reference in their entireties,and from which priority is claimed.

GRANT INFORMATION

This invention was made with government support under Grant No. U19A1068021 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

1. INTRODUCTION

The present invention relates to methods for reducing and/or delayingone or more signs of aging and/or degeneration which compriseadministering, to a subject in need of such treatment, one or more ofthe compounds as set forth herein.

2. BACKGROUND OF THE INVENTION 2.1 Aging

It is estimated that in the next 25 years, the number of individualsover the age of 65 in the United States will double (U.S. Department ofHealth and Human Services, 2003). With increased chronological age,there is progressive attrition of homeostatic reserve of all organsystems (Resnick and Dosa, 2004). As a consequence, aged individualshave a dramatically increased risk of numerous debilitating diseasesincluding bone fractures, cardiovascular disease, cognitive impairment,diabetes and cancer (Resnick and Dosa, 2004). Therefore, as Americandemographics shift, increasing demands are placed on our health caresystem (Crippen, 2000). Identifying strategies to prevent or delayage-associated frailty and diseases is imperative for maintaining thehealth of our population as well as our nation's economy.

The molecular basis of the progressive loss of homeostatic reserve withaging is controversial (Kirkwood, 2005b; Resnick and Dosa, 2004). Thereis strong evidence that genetics contribute significantly to lifespanand end-of-life fitness (Hekimi and Guarente, 2003). This wasdemonstrated by identifying single genes that when mutated oroverexpressed attenuate and extend lifespan, respectively (Kurosu etal., 2005). Many of the genes that regulate lifespan affect the growthhormone (GH)/insulin-like growth factor 1 (IGF1) axis, which controlscellular proliferation and growth (Kenyon, 2005). Suppression of thisaxis extends lifespan significantly and delays age-related diseases(Bartke, 2005).

Alternatively, the disposable soma theory of aging posits that aging isthe consequence of accumulation of stochastic molecular and cellulardamage (Kirkwood, 2005b). The precise nature of the damage that isresponsible for aging-related degenerative changes remains ill-defined,but may include mitochondrial damage, telomere attrition, nucleardysmorphology, accumulation of genetic mutations, DNA, protein ormembrane damage.

There are several lines of evidence to support the notion that DNAdamage is one type of molecular damage that contributes to aging. At theforefront of this is the observation that the majority of humanprogerias (or syndromes of accelerated aging) are caused by inheritedmutations in genes required for genome maintenance, including Wernersyndrome, Cockayne syndrome, trichothiodystrophy and ataxiatelangiectasia (Hasty et al., 2003). Furthermore both DNA lesions(Hamilton et al., 2001) and genetic mutations caused by DNA damage(Dolle et al., 2002) accumulate in tissues with aging. Finally, miceharboring germ-line mutations that confer resistance to genotoxic stressare long-lived (Maier et al., 2004; Migliaccio et al., 1999).

ERCC1-XPF is a highly conserved structure-specific endonuclease that isrequired for at least two DNA repair mechanisms in mammalian cells:nucleotide excision repair (Sijbers et al., 1996) and DNA interstrandcrosslink repair (Niedernhofer et al., 2004). Genetic deletion of eitherErcc1 or Xpf in the mouse causes an identical and very severe phenotype(McWhir et al., 1993; Tian et al., 2004; Weeda et al., 1997). Embryonicdevelopment of null mice is normal, but postnatally they developnumerous symptoms associated with advanced age including epidermalatrophy and hyperpigmentation, visual impairment, cerebral atrophy withcognitive deficits, cerebellar degeneration, hypertension, renalinsufficiency, decreased liver function, anemia and bone marrowdegeneration, osteoporosis, sarcopenia, cachexia, and decreased lifespan(Niedernhofer et al., 2006; Prasher et al., 2005; Weeda et al., 1997,and see International Patent Application Publication No. WO2006/052136).

To determine if this progeroid phenotype had commonalities with thenatural aging process, the transcriptome from the liver of Ercc1^(−/−)mice was compared to that of old wild type mice and a highly significantcorrelation was identified (Niedernhofer et al., 2006). Similarexpression changes were also identified in young wild type mice afterchronic exposure to a DNA damaging agent. This provides directexperimental evidence that DNA damage induces changes that mimic agingat the fundamental level of gene expression.

Gene ontology classification of the expression data was used to predictpathophysiologic changes that were similar in Ercc1^(−/−) mice and oldwild type mice (Niedemhofer et al., 2006). These predictions were testedcomparing Ercc1^(−/−) mice, young and old wild type mice. For allpredictions tested, Ercc1^(−/−) were more similar to old mice than totheir wild type littermates despite the vast difference in age (3 weeksvs. 120 weeks). Both Ercc1^(−/−) and old mice displayedhyposomatotropism, hepatic accumulation of glycogen and triglycerides,decreased bone density, increased peroxisome biogenesis, increasedapoptosis and decreased cellular proliferation. Therefore, Ercc1^(−/−)and old mice share not only broad changes in gene expression, but alsoendocrine, metabolic and cell signaling changes. This implies thatERCC1-deficient mice are an accurate and rapid model system for studyingsystemic aging in mammals. A case of human progeria caused by ERCC1-XPFdeficiency with symptoms near-identical to those observed inERCC1-deficient mice has been reported (Niedernhofer et al., 2006).Therefore function of ERCC1-XPF is conserved from man to mouse and thediscovery of what is driving aging-like degenerative changes inERCC1-deficient mice will have direct implications for human health.

A number of the degenerative changes associated with normal aging may bemanifested in an accelerated form and/or in younger individuals.Examples of such degenerative disorders include neurodegenerativedisorders such as Alzheimer's disease, Huntington's disease, Parkinson'sdisease and osteoporosis, and joint degenerative conditions such asosteoarthritis, rheumatoid arthritis and intervertebral discdegeneration.

2.2 Free Radicals, Aging and Degeneration

Cells undergo some degree of oxidative stress by way of generatingreactive oxygen species (“ROS”) and reactive nitrogen species (“RNS”).Specifically, the cellular respiration pathway generates ROS and RNSwithin the mitochondrial membrane of the cell (Kelso et al., 2001).Reactive oxygen species include free radicals, reactive anionscontaining oxygen atoms, and molecules containing oxygen atoms that caneither produce free radicals or are chemically activated by them.Specific examples include superoxide anion, hydroxyl radical, andhydroperoxides.

Naturally occurring enzymes, such as superoxide dismutase (“SOD”) andcatalase, detoxify ROS and RNS radicals to allow normal metabolicactivity to occur. Significant deviations from cell homeostasis, such ashemorrhagic shock, lead to an oxidative stress state, thereby causing“electron leakage” from the mitochondrial membrane. This “electronleakage” produces an excess amount of ROS for which the cell's naturalantioxidants cannot compensate. Specifically, SOD cannot accommodate theexcess production of ROS associated with hemorrhagic shock whichultimately leads to premature mitochondria dysfunction and cell deathvia apoptosis (Kentner et al., 2002).

Cardiolipin (“CL”) is an anionic phospholipid exclusively found in theinner mitochondrial membrane of eukaryotic cells (Iverson and Orrenius,2002). Under normal conditions, the pro-apoptotic protein cytochrome Cis anchored to the mitochondrial inner membrane by binding with CL(Tuominen, et al., 2002). The acyl moieties of CL are susceptible toperoxidation by reactive oxygen species. When ROS are generated withinmitochondria in excess quantities, cytochrome C bound to CL can functionas an oxidase and induces extensive peroxidation of CL in themitochondrial membrane (Kagan et al., 2005a and 2005b).

The peroxidation of the CL weakens the binding between the CL andcytochrome

C (Shidoji, et al., 1999). This leads to the release of the cytochrome Cinto the mitochondrial intermembrane space, inducing apoptotic celldeath. Further, the peroxidation of CL has the effect of opening themitochondrial permeability transition pore (“MPTP”; Dolder et al., 2001;Imai et al., 2003). Accordingly, the mitochondrial membrane swells andreleases the cytochrome C into the cytosol. Excess cytochrome C in thecytosol leads to cellular apoptosis (Iverson et al., 2003).

Moreover, mitochondrial dysfunction and cell death may ultimately leadto multiple organ failure despite resuscitative efforts or supplementaloxygen supply (Cairns, 2001). Reduction of oxidative stress delays, eveninhibits, physiological conditions that otherwise might occur, such ashypoxia.

One of the limitations of SOD is that it cannot easily penetrate thecell membrane. However, nitroxide radicals, such as TEMPO(2,2,6,6-tetramethylpiperidine-N-oxyl) and its derivatives, have beenshown to penetrate the cell membrane better than SOD and inhibit theformation of ROS, particularly superoxide, due to their reduction by themitochondrial electron transport chain to hydroxyl amine radicalscavengers (Wipf et al., 2005a).

Examples of antioxidant agents include agents set forth in US 2007161544and US2007161573, such as, for example, XJB-5-131.

The aging-related and degenerative changes described above areassociated with deterioration in the context of impaired regenerativecapacity. There appears to be an inverse relationship between themaximum lifespan of a species and the amount of ROS and RNS that speciesproduces (Finkel, 2000). Caloric restriction, which reduces ROS and RNSproduction, promotes longevity and delays the onset of age-relateddiseases (Heilbronn, 2003). Thus, effective ROS and RNS scavengers arepotential therapeutic agents for age-related pathologies anddegenerative conditions.

3. SUMMARY OF THE INVENTION

The present invention relates to methods for reducing and/or delayingone or more signs of aging and/or the progression of a degenerativedisorder comprising administering, to a subject in need of suchtreatment, a compound as disclosed herein according to Formula 1, 2 or3, below, including, but not limited to, a compound which comprises aTEMPO or 4-amino-TEMPOL functional group, such as, but not limited to,XJB-5-131, JP4-039, JED-E71-37 or JED-E71-58 (see FIG. 1A-D for chemicalstructures of these latter compounds). The invention is based, at leastin part, on (i) the discovery that treatment with the 4-amino-TEMPOLcontaining compound XJB-5-131 inhibited the development of variousindicia of senescence and degeneration in vivo in a murine model ofaging, Ercc1^(−/−) mice; (ii) the discovery that 4-amino-TEMPOLcontaining JP4-039 reduced cellular senescence in vitro (iii) thediscovery that two compounds which each contain two 4-amino-TEMPOLgroups, JED-E71-37 and JED-E71-58, reduced oxidation-related cell damagein vitro and epidermal atrophy in vivo; and (iv) the discovery thatXJB-5-131 delayed progression of symptoms in an animal model ofHuntington's disease.

4. BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A-D. Structures of (A) XJB-5-131; (B) JP4-039; (C) JED-E71-37; and(D) JED-E71-58.

FIG. 2A-D. Compounds as set forth herein. (A) Non-limiting examples ofcertain nitroxides. The logP values were estimated using the onlinecalculator of molecular properties and drug likeness on theMolinspirations Web site (www.molinspiration.com/cgi-bin/properties).TIPNO is “tert-butyl isopropyl phenyl nitroxide.” (B) Examples ofstructures of certain compounds as set forth herein, and the structureof TEMPOL. (C) Example of a synthetic pathway for theTEMPO-hemigramicidin conjugates. (D) Schematic of a synthesis protocolfor JP4-039.

FIG. 3. Schematic diagram of mErcc1 targeting constructs. In theknock-out allele (−) exon 7 was interrupted with a neomycin selectablemarker. In the hypomorphic allele (Δ), a 7 amino acid deletion wasplaced at the C-terminus of the protein to humanize the gene and a neocassette in intron 9, which attenuated expression of Ercc 1. In theconditional allele (cond), exon 7 was fused with the cDNA of exons 8-10and the entire region foxed with loxP sites recognized byCre-recombinase, allowing tissue-specific deletion of ERCC1, for examplein the skin (see FIG. 10).

FIG. 4. Immunodetection of XPF, the obligate binding partner of ERCC1,in protein extracts isolated from the liver of Ercc1 mutant mice. Thegenotype of the mice is indicated above the lanes. Below the blots areindicated the calculated level of XPF expression in the Ercc1 mutantmice, relative to wild type (100%).

FIG. 5. Clonogenic survival assay measuring the sensitivity ofERCC1-deficient cells to the DNA damaging agent mitomycin C. Wild-typedata is represented by a circle; Ercc1^(−/↓) by a square; Ercc1^(−/Δ) bya triangle, and Ercc1^(−/−) by an open square.

FIG. 6A-D. (A) Lifespan of mice expressing various levels of ERCC1-XPFcompared to wild type C57B1/6 mice (100%; adapted from (Rowlatt et al.,1976). Wild-type data is represented by a diamond; Ercc1^(−/↓) by asquare; Ercc1^(−/Δ) by a triangle, and Erccl^(−/−) by crosses. (B) Thespontaneous premature aging phenotype of Ercc1^(−/Δ) mice. The mice werehealthy for the first 8-9 weeks of life then began to displayprogressive symptoms associated with aging. These included dystonia,trembling, weight loss, proximal muscle wasting, kyphosis, ataxia, lossof vision and hearing, impaired kidney and liver function and urinaryincontinence.

Histopathologic analysis and microCT revealed osteoporosis,intervertebral disc degeneration, bone marrow hypoplasia, epidermalatrophy and neurodegeneration. The maximum lifespan was seven months.(C) Clinical features of progeria caused by mutation of XPF andprogeroid mice due to mutation of Ercc1. (D) Age at onset of progeroidsymptoms in Ercc1^(−/Δ) mice as determined from a cumulative history ofexamining >60 mice. Symptoms marked with an asterisk indicate symptomscaused by neurodegeneration.

FIG. 7. Experimental system for determining the impact of a therapeuticcompound on the healthspan of Ercc1^(−/Δ) mice with accelerated aging.Transgenic mice or mutant animals were born from heterozygous crossings.Only litters with two Ercc1^(−/Δ) mice were enrolled in the study sothat siblings could be used to assess the effect of a therapeuticcompound, XJB-5-131 vs. vehicle in a pair-wise comparison. Treatmentswere initiated before the mice became symptomatic. The age at onset ofprogeroid symptoms were measured biweekly by an investigator blinded tothe treatment of the mice.

FIG. 8. Summary table showing the age at onset of progeroid symptoms inErcc1^(−/Δ) mice treated with XJB-5-131 or vehicle only. TreatingErcc1^(−/Δ) mice with one example of the compounds as disclosed herein,XJB-5-131, delayed the onset of progeroid symptoms and agingpathologies. Values marked with an asterisk indicate a symptom ofaccelerated aging that was significantly delayed (including dystonia,ataxia, muscle wasting and decreased spontaneous activity or lethargy).The aging score reflected the relative age at onset of all progeroid 3 0symptoms in a mouse treated with XJB-5-131 vs. its sibling treated withoil only and was thus a measure of healthspan.

FIG. 9A-D. (A) Comparison of sibling Ercc1^(−/Δ) mice treated withXJB-5-131 or vehicle only (sunflower seed oil) according to the protocolshown in FIG. 7. The duration of treatment of mice in this figure wasthree times per week beginning at 5 weeks of age and continuingthroughout their lifespan. The mouse treated with XJB-5-131 had less:(1) neurodegeneration seen as a normal reflex (splaying of the hindlimbs rather than clasping) upon tail suspension; (2) muscle wasting inthe hind quarters and (3) improved general appearance (bottom panel).(B) Bar graph showing glycosaminoglycan content of intervertebral discsof Ercc1^(−/Δ) mice either treated with XJB-5-131 or vehicle (sunflowerseed oil) according to the protocol shown in FIG. 7. The duration oftreatment of mice in this figure was three times per week beginning at 5weeks of age and continuing throughout their lifespan. (C) Decreasedneurodegeneration in the cerebellum of a mouse treated with XJB-5-131compared to a sibling Ercc1^(−/Δ) mouse that was treated with vehicleonly (oil). Neurodegeneration was detected by immunostaining for glialacidic fibrillary protein (GFAP; brown staining). (D) Preservation ofinsulin-producing β-islet cells (patches of pale purple highlighted witharrows) in the pancreas of an XJB-5-131-treated mouse. Pancreaticsections were stained with haematoxylin and eosin.

FIG. 10A-D. (A) Effects of (photo)aging in Ercc1^(−/Δ) mice eithertreated with XJB-5-131 (80 μg emulsified in a topical cream) or creamonly according to the protocol shown in FIG. 7. The duration oftreatment of mice in this figure was daily for five days post-UVirradiation. (B) Compounds as disclosed herein protect skin fromphotoaging by improving keratin production. Skin ofErcc1^(−/cond);K14-Cre mice (missing ERCC1-XPF expression only in theskin) were treated with 20 J/m2 (half of the minimal erythemal dose)five times per week for 10 weeks to induce photoaging of the skin.Immediately after UV exposure, XJB-5-131 was applied (80 μg in 50 μL ofcream) using a cotton applicator. Twenty-four hours after the lasttreatment, mice were euthanized and skin harvested for histopathologicexamination. Skin sections were immunostained for keratin (red), DNA toreveal nuclei (blue) and actin (green). (C) Compounds as disclosedherein protect skin from inflammation in response to ultravioletradiation. Skin of Ercc 1^(−/cond);K14-Cre mice (missing ERCC1-XPFexpression only in the skin) were treated with 500 J/m2 (12.5× theminimal erythemal dose) to induce erythema. Immediately after UVexposure, XJB-5-131 was applied (80 μg in 50 μL of cream) using a cottonapplicator. Twenty-four hours after treatment, mice were euthanized andskin harvested for histopathologic examination. Skin sections wereimmunostained for keratin (red), DNA to reveal nuclei (blue) and actin(green). The thickness of the epidermal layer (indicated by the whitebracket) is less in mice treated with XJB-5-131 than in mice treatedwith cream alone. (D) Compounds as disclosed herein reduce epidermalatrophy. Old (>24 months) normal mice were shaved and their skin treatedwith JP4-039 (450 μg in 50 μL of cream) or vehicle (cream) only fivetimes per week for 10 weeks. Twenty-four hours after the last treatment,mice were euthanized and skin harvested for histopathologic examination.Skin sections were stained for DNA to reveal nuclei (blue) and actin(green). The thickness of the epidermal layer (indicated by the bracket)was significantly greater in mice treated with JP4-039 than in micetreated with vehicle (cream) alone, demonstrating preservation of skinthickness or reversal of epidermal atrophy, a common age-associatedfeature.

FIG. 11A-B. Weights as a function of age of (A) male and (B) femaleErcc1^(−/Δ) mice either treated with XJB-5-131 or vehicle (sunflowerseed oil) according to the protocol shown in FIG. 7.

FIG. 12. SA-β galactosidase (a marker of cellular senescence) stainingin mouse embryonic fibroblast (“MEF”) cells prepared from Ercc1^(−/−)mice, where the MEF cells were either treated with XJB-5-131 (500 μmdissolved in media) or media alone continuously for 48 hours prior tofixing and staining the cells.

FIG. 13A-B. γH2AX immunostaining of Ercc1^(−/−) primary MEF grown at 20%oxygen to induce oxidative stress. Phosphorylated H2AX (γH2AX is amarker of cellular senescence and DNA damage. The MEF cells were eithertreated with XJB-5-131 (A) or E71-58 (B), fewer cells stained positivelyfor γH2AX.

FIG. 14. Apoptosis in MEF cells prepared from Ercc1^(−/−) mice, wherethe MEF cells were either treated with XJB-5-131 (500 nM dissolved inmedia) or media alone continuously for 48 hours prior to fixing andstaining the cells.

FIG. 15. Effects of varying doses of 24-039, on proliferation and growthof MEF cells prepared from Ercc1^(−/−) mice. JP4-039 was not toxic tocells at concentrations as high as 10 μM.

FIG. 16. Effects of varying doses of JP4-039, on proliferation andgrowth of MEF cells prepared from wild-type mice.

FIG. 17. Levels of p16, a marker of irreversible cellular senescence, inMEF cells prepared from Ercc1^(−/−) mice, where the MEF cells wereeither treated with JP4-039 (10 μM dissolved in media) or media alonefor 48 hours prior to fixing and immunostaining of cells.

FIG. 18. Cell proliferation of primary MEF cells prepared fromErcc1^(−/−) mice and grown in conditions of oxidative stress, where theMEF cells were either treated with JED-E71-37, JED-E71-58, (91 μMdissolved in media) or media alone for 48 hours prior to fixing andstaining the cells.

FIG. 19. XJB-5-131 preserved motor function in a mouse model ofHuntington's disease. Five R6/2 mice, which model Huntington's disease,were treated three times per week with 2 mg/kg XJB-5-131 beginning at 5weeks of life according to the scheme in FIG. 7. Motor function wasassessed weekly by measuring the time animals were able to remainbalanced on a rotarod, according to standard methods. Mice treated withXJB-5-131 performed significantly better at six and seven weeks of age,demonstrating that XJB-5-131 delayed the progression of symptoms ofHuntington's disease.

5. DETAILED DESCRIPTION OF THE INVENTION

For clarity of presentation, and not by way of limitation, the detaileddescription of the invention is divided into the following subsections:

-   -   (i) compounds;    -   (ii) signs of aging that may be modulated;        and    -   (iii) methods of treatment.

5.1 Compounds

The present invention provides for the use of any of a number ofcompounds as set forth herein, used alone or in combination. The presentinvention further provides for pharmaceutical compositions comprisingsaid compounds, where a pharmaceutical composition comprises an amountof a compound effective in reducing and/or delaying one sign of agingand/or degeneration, in a suitable pharmaceutical carrier.

Whilst we are not bound by any theory, the compounds set forth hereininclude antioxidant compounds, free radical scavengers, and inparticular, mitochondrial targeted free radical scavengers. A compoundas disclosed herein according to Formula 1, 2, or 3, below, includes,but is not limited to, a compound which comprises a TEMPO or4-amino-TEMPOL functional group, such as, but not limited to, XJB-5-131,JP4-039, JED-E71-37 or JED-E71-58.

An antioxidant compound as that term is used herein, is a compound thatdecreases the rate of oxidation of another compound or that inhibits orprevents reaction between a substance and oxygen or an oxygen containingcompound. A compound may be determined to be an antioxidant compound byassessing its ability to decrease molecular oxidation and/or cellularsequellae of oxidative stress, for example, but not by way oflimitation, the ability to decrease lipid peroxidation and/or decreaseoxidative damage to protein or nucleic acid. Preferably, an antioxidantof the invention has a level of antioxidant activity between 0.01 and1000 times the antioxidant activity of ascorbic acid in at least oneassay that measures antioxidant activity.

Free radical scavengers used herein are compounds that react with freeradicals. Free radical scavengers reduce free radical-induced damage,and protect against the indirect effects of free radicals produced byionizing radiation, etc.

In one non-limiting embodiment, the compound has the structure:

wherein X is one of

andR₁, R₂ and R₄ are, independently, C₁-C₆ straight or branched-chainalkyl, optionally including a phenyl (C₆H₅) group, that optionally ismethyl-, hydroxyl- or fluoro-substituted, including including: methyl,ethyl, propyl, 2-propyl, butyl, t-butyl, pentyl, hexyl, benzyl,hydroxybenzyl (e.g., 4-hydroxybenzyl), phenyl and hydroxyphenyl. R₃ is—NH—R₅, —O—R₅ or —CH₂—R₅, and R₅ is an —N—O. or —N—OH containing group.R is —C(O)—R₆ or —C(O)O—R₆, and R₆ is C₁-C₆ straight or branched-chainalkyl optionally comprising one or more phenyl (—C₆H₅) groups, and thatoptionally are methyl-, ethyl-, hydroxyl- or fluoro-substituted,including Boc (R═—C(O)O-tert-butyl) and Cbz (R═—C(O)O-benzyl (Bn))groups. Excluded from this is the enantiomer XJB-5-208. As used herein,unless indicated otherwise, for instance in a structure, all compoundsand/or structures described herein comprise all possible stereoisomers,individually or mixtures thereof.

As indicated above, R₅ is an —N—O. or —N—OH containing group. As isknown to one ordinarily skilled in the art, nitroxide and nitroxidederivatives, including TEMPOL and associated TEMPO derivatives arestable radicals that can withstand biological environments. Therefore,the presence of the 4-amino-TEMPO, TEMPOL or another nitroxide “payload”within the mitochondria membrane can serve as an effective and efficientelectron scavenger of the ROS being produced within the membrane.Non-limiting examples of this include TEMPO(2,2,6,6-Tetramethyl-4-piperindine 1-oxyl) and TEMPOL (4-Hydroxy-TEMPO),in which, when incorporated into the compound described herein, form,for example, when R₃ is —NH—R₅, —O—R₅:

Additional non-limiting examples of —N—O. or —N—OH containing group areprovided in Table 1 and in FIG. 2A (from Jiang, et al., 2007). A personof ordinary skill in the art would be able to conjugate (covalentlyattach) any of these compounds to the rest of the compound using commonlinkers and/or conjugation chemistries, such as the chemistriesdescribed herein. Table 1 provides a non-limiting excerpt from a list ofover 300 identified commercially-available —N—O. or —N—OH containingcompounds that may be useful in preparation of the compounds orcompositions described herein.

TABLE 1 Commercially-available N—O• or N═O containing groups StructureName CAS No.

Trimethylamine N-Oxide 1184-78-7

N,N-Dimethyldodecylamine N-Oxide 1643-20-5 70592-80-2

N-Benzoyl-N-Phenylhydroxylamine 304-88-1

N,N-Diethylhydroxylamine 3710-84-7

N,N-Dibenzylhydroxylamine 14165-27-6 621-07-8

Di-Tert-Butyl Nitroxide 2406-25-9

N,N- Dimethylhydroxylamine Hydrochloride 16645-06-0

Metobromuron 3060-89-7

Benzyl-Di-Beta-Hydroxy Ethylamine-N-Oxide

Bis(Trifluoromethyl)Nitroxide 2154-71-4

Triethylamine N-Oxide 2687-45-8

N-Metboxy-N- Methylcarbamate 6919-62-6

N,N-BIS(2-CHLORO-6- FLUOROBENZYL)-N- [(([2,2-DICHLORO-1-(1,4-THIAZINAN-4-YL+) ETHYLIDENE]AMINO) CARBONYL)OXY]AMINE

Tri-N-Octylamine N-Oxide 13103-04-3

DIETHYL (N-METHOXY-N- METHYLCARBAMOYLMETHYL) PHOSPHONATE 124931-12-0

N-Methoxy-N-Methyl-2- (Triphenylphosphoranylidene) Acetamide 129986-67-0

N-Methoxy-N-Methyl-N′- [5-Oxo-2- (Trifluoromethyl)-5h-Chromeno[2,3-B]Pyridi+ N-3-Yl]Urea

N-[(4-Chlorobenzyl)Oxy] - N-([5-Oxo-2-Phenyl-1,3- Oxazol-4(5h)-Yliden]Methyl+) Acetamide

N-Methylfurohydroxamic Acid 109531-96-6

N,N-Dimethylnonylamine N-Oxide 2536-13-2

N-(Tert-Butoxycarbonyl)-L- Alanine N′-Methoxy-N′- Methylamide 87694-49-3

1-(4-Bromophenyl)-3- (Methyl([3- (Trifluoromethyl)Benzoyl]Oxy)Amino)-2-Prop+ En-1-One

2- ([[(Anilinocarbonyl)Oxy] (Methyl)Amino]Methylene)-5-(4-Chlorophenyl)-1,3+- Cyclohexanedione

N-Methoxy-N-Methyl-2- (Trifluoromethyl)-1,8- Naphthyridine-3-Carboxamide

N-Methoxy-N-Methyl- Indole-6-Carboxamide

Desferrioxamin

AKOS 91254 127408-31-5

N-[(3s, 4r)-6-Cyano-3,4- Dihydro-3-Hydroxy-2,2-Dimethyl-2h-1-Benzopyran- 4-Y+ L]-N-Hydroxyacetamide 127408-31-5

N-Methoxy-N-Methyl-1,2- Dihydro-4-Oxo- Pyrrolo[3,2,1-Ij]Quinoline-5-Carboxa+ Mide

Fr-900098

2,2′-(Hydroxyimino)Bis- Ethanesulfonic Acid Disodium Salt 133986-51-3

Fmoc-N-Ethyl- Hydroxylamine

Bis(N,N- Dimethylhydroxamido) Hydroxooxovanadate

Pyraclostrobin 175013-18-0

1-Boc-5-Chloro-3- (Methoxy-Methyl- Carbamoyl)Indazole

N-Methoxy-N-Methyl- Thiazole-2-Carboxamide

4,4-Difluoro-N-Methyl-N- Methoxy-L-Prolinamide Hcl

3-Fluoro-4- (Methoxy(Methyl)Carbamoyl) Phenylboronic Acid 913835-59-3

1-Isopropyl-N-Methoxy-N- Methyl-1h- Benzo[D][1,2,3]Triazole-6-Carboxamide 467235-06-9

(Trans)-2-(4-Chlorophenyl)- N-Methoxy-N- Methylcyclopropanecarboxamide

Bicyclo[2.2.1]Heptane-2- Carboxylic Acid Methoxy- Methyl-Amide

Akos Bc-0582

3-(N,O- Dimethylhydroxylaminocarbonyl) Phenylboronic Acid, Pinacol Ester

1 -Triisopropylsilanyl-1h- Pyrrolo[2,3-B]Pyridine-5- Carboxylic AcidMethoxy+- Methyl-AmideAccording to one embodiment, the compound has the structure

or the structure

Wherein R is —NH—R₁, —O—R₁ or —CH₂—R₁, and R₁ is an —N—O. or —N—OHcontaining group. In one embodiment, R is —NH—R₁, and in another R isN-TEMPO.

In one non-limiting embodiment, the compound has the structure:

In which R1, R2 and R3 are, independently, C₁-C₆ straight orbranched-chain alkyl, optionally including a phenyl (C₆H₅) group, thatoptionally is methyl-, hydroxyl- or fluoro-substituted, including2-methyl propyl, benzyl, methyl-, hydroxyl- or fluoro-substitutedbenzyl, such as 4-hydroxybenzyl. R4 is an —N—O. or —N—OH containinggroup. R is —C(O)—R5 or —C(O)O—R5, and R5 is C₁-C₆ straight orbranched-chain alkyl, optionally comprising one or more phenyl (—C₆H₅)groups, and that optionally are methyl-, ethyl-, hydroxyl- orfluoro-substituted, including Boc and Cbz groups. In certain specificembodiments, in which R4 is TEMPO, the compound has one of thestructures A, A1, A2, or A3 (Ac=Acetyl=CH₃C(O)—):

In another non-limiting embodiment, the compound has the structure

In which R1, R2 and R3 are, independently, C₁-C₆ straight orbranched-chain alkyl, optionally including a phenyl (C₆H₅) group, thatoptionally is methyl-, hydroxyl- or fluoro-substituted, including2-methyl propyl, benzyl, methyl-, hydroxyl- or fluoro-substitutedbenzyl, such as 4-hydroxybenzyl. R4 is an —N—O. or —N—OH containinggroup. R is —C(O)—R5 or —C(O)O—R5, and R5 is C₁-C₆ straight orbranched-chain alkyl, optionally comprising one or more phenyl (—C₆H₅)groups, and that optionally are methyl-, ethyl-, hydroxyl- orfluoro-substituted, including Boc and Cbz groups. In certain specificembodiments, in which R4 is TEMPO, the compound has one of thestructures D, D1, D2, or D3 (Ac=Acetyl=CH₃C(O)—):

The compounds described above, such as the compound of Formula 1, can besynthesized by any useful method. The compound JP4-039 may besynthesized, for example and not by limitation, by the method depictedin FIG. 2D. In one embodiment, a method of making the compounds offormula 1 is provided, and the compounds are synthesized by thefollowing steps:

-   A. reacting an aldehyde of structure R₁—C(O)—, wherein, for example    and without limitation, R₁ is C₁-C₆ straight or branched-chain    alkyl, optionally including a phenyl (C₆H₅) group, that optionally    is methyl-, hydroxyl- or fluoro-substituted, including including:    methyl, ethyl, propyl, 2-propyl, butyl, t-butyl, pentyl, hexyl,    benzyl, hydroxybenzyl (e.g., 4-hydroxybenzyl), phenyl and    hydroxyphenyl, with (R)-2-methylpropane-2-sulfinamide to form an    imine, for example

-   B. reacting a terminal alkyne-1-ol (CHC—R₂—C—OH), wherein, for    example and without limitation, R₂ is not present or is branched or    straight-chained alkylene, including methyl, ethyl, propyl, etc.,    with a tert-butyl)diphenylsilane salt to produce a    t-butyldiphenyl(alkylynyloxy)silane, for example

-   C. reacting (by hydrozirconation) the    t-butyldiphenyl(alkylynyloxy)silane with the imine in the presence    of an organozirconium catalyst to produce a    (t-butyldiphenylsilyloxy)alkylenyl amine hydrochloride, for example

-   D. acylating the (t-butyldiphenylsilyloxy)alkylenyl amine    hydrochloride to produce a (t-butyldiphenylsilyloxy)alkylenyl    carbamate, for example

wherein, for example and without limitation, R₃ is C₁-C₆ straight orbranched-chain alkyl, optionally including a phenyl (C₆H₅) group, thatoptionally is methyl-, hydroxyl- or fluoro-substituted, includingincluding: methyl, ethyl, propyl, 2-propyl, butyl, t-butyl, pentyl,hexyl, benzyl, hydroxybenzyl (e.g., 4-hydroxybenzyl), phenyl andhydroxyphenyl;

-   E. removing the t-butyldiphenylsilyl group from the    (t-butyldiphenylsilyloxy)alkylenyl carbamate to produce an alkylenyl    carbamate-1-ol, for example

-   F. oxidizing the alkylenyl carbamate-1-ol to produce an alkylenyl    carbamate-1-carboxylic acid, for example

and

-   G. reacting alkylenyl carbamate-1-carboxylic acid with a    nitroxide-containing compound comprising one of a hydroxyl or amine    in a condensation reaction to produce the compound as set forth    herein, for example

Wherein R₄ is —NH—R₄ or —O—R₄, and R₄ is an —N—O. or —N—OH containinggroup, such as described above.

In another non-limiting embodiment, a compound is provided having thestructure R1-R2-R3 in which R1 and R3 are a group having the structure—R4-R5, in which R4 is a mitochondria targeting group and R5 is —NH—R6,—O—R6 or —CH₂—R6, wherein R6 is an —N—O. or —N—OH containing group, suchas TEMPO. R1 and R2 may be the same or different. Likewise, R4 and R5for each of R1 and R3 may be the same or different. R2 is a linker that,in one non-limiting embodiment, is symmetrical. FIGS. 1C and 1D depicttwo examples of such compounds. In one embodiment, R1 and R2 have thestructure shown in formulas 1, 2, or 3, above, with all groups asdefined above, including structures A, A1, A2 A3, D, D1, D2 and D3,above, an example of which is compound JED-E71-58, shown in FIG. 1D. Inanother embodiment, R1 and R2 are, independently, a gramicidinderivative, for example, as in the compound JED-E71-37, shown in FIG.1C. Examples of gramicidin derivatives are provided herein, such asXJB-5-131 and XJB-5-125 (see FIG. 2B), and are further described bothstructurally and functionally in United States Patent Publication Nos.20070161573 and 20070161544 as well as in Jiang, et aL 2007; Hoye, etal., 2008; and Wipf, et al., 2005a. The XJB compounds can be linked intoa dimer, for example and without limitation, by reaction with thenitrogen of the BocHN groups (e.g., as in XJB-5-131), or with an amine,if present, for instance, if one or more amine groups of the compound isnot acylated to form an amide (such as NHBoc or NHCbx).

In Jiang, et al., with a model of ActD-induced apoptosis in mouseembryonic cells, a library of nitroxides were screened to explorestructure-activity relationships between their antioxidant/antiapoptoticproperties and chemical composition and three-dimensional (3D)structure. High hydrophobicity and effective mitochondrial integrationwere deemed necessary but not sufficient for highantiapoptotic/antioxidant activity of a nitroxide conjugate. Bydesigning conformationally preorganized peptidyl nitroxide conjugatesand characterizing their 3D structure experimentally (circular dichroismand NMR) and theoretically (molecular dynamics), they established thatthe presence of the β-turn/β-sheet secondary structure is essential forthe desired activity. Monte Carlo simulations in model lipid membranesconfirmed that the conservation of the D-Phe-Pro reverse turn in hemi-GSanalogs ensures the specific positioning of the nitroxide moiety at themitochondrial membrane interface and maximizes their protective effects.These insights into the structure-activity relationships ofnitroxide-peptide and -peptide isostere conjugates are helpful in thedevelopment of new mechanism-based therapeutically effective agents,such as those described herein.

Targeting group R4 may be a membrane active peptide fragment derivedfrom an antibiotic molecule that acts by targeting the bacterial cellwall. Examples of such antibiotics include: bacitracins, gramicidins,valinomycins, enniatins, alamethicins, beauvericin, serratomolide,sporidesmolide, tyrocidins, polymyxins, monamycins, and lissoclinumpeptides. The membrane-active peptide fragment derived from anantibiotic may include the complete antibiotic polypeptide, or portionsthereof having membrane, and preferably mitochondria-targetingabilities, which is readily determined, for example, by cellularpartitioning experiments using radiolabled peptides. Examples of usefulgramicidin-derived membrane active peptide fragments are theLeu-D-Phe-Pro-Val-Orn and D-Phe-Pro-Val-Orn-Leu hemigramicidinfragments. As gramicidin is circular, any hemigramicidin 5-mer isexpected to be useful as a membrane active peptide fragment, includingLeu-D-Phe-Pro-Val-Orn, D-Phe-Pro-Val-Orn-Leu, Pro-Val-Orn-Leu-D-Phe,Val-Orn-Leu-D-Phe-Pro and Orn-Leu-D-Phe-Pro-Val (from Gramicidin S). Anylarger or smaller fragment of gramicidin, or even larger fragmentscontaining repeated gramicidin sequences (e.g.,Leu-D-Phe-Pro-Val-Orn-Leu-D-Phe-Pro-Val-Orn-Leu-D-Phe-Pro) are expectedto be useful for membrane targeting, and can readily tested for suchactivity. In one embodiment, the Gramicidin S-derived peptide comprisesa β-turn, which appears to confer to the peptide a high affinity formitochondria. Derivatives of Gramicidin, or other antibiotic fragments,include isosteres (molecules or ions with the same number of atoms andthe same number of valence electrons—as a result, they can exhibitsimilar pharmacokinetic and pharmacodynamic properties), such as(E)-alkene isosteres (see, United States Patent Publication Nos.20070161573 and 20070161544 for exemplary synthesis methods). As withGramicidin, the structure (amino acid sequence) of bacitracins, othergramicidins, valinomycins, enniatins, alamethicins, beauvericin,serratomolide, sporidesmolide, tyrocidins, polymyxins, monamycins, andlissoclinum peptides are all known, and fragments of these can bereadily prepared and their membrane-targeting abilities can easily beconfirmed by a person of ordinary skill in the art.

Alkene isosteres such as (E)-alkene isosteres of Gramicidin S (i.e.,hemigramicidin) were used as part of the targeting sequence. See FIG. 2Cfor a synthetic pathway for (E)-alkene isosteres and reference number 2for the corresponding chemical structure. First, hydrozirconation ofalkyne (FIG. 2C, compound 1) with Cp₂ZrHCl is followed bytransmetalation to Me₂Zn and the addition of N-Boc-isovaleraldimine. Theresulting compound was then worked up using a solution oftetrabutylammonium fluoride (“TBAF”) and diethyl ether with a 74% yield.The resulting compound was then treated with acetic anhydride,triethylamine (TEA), and 4-N,N¹-(dimethylamino)pyridine (“DMAP”) toprovide a mixture of diastereomeric allylic amides with a 94% yieldwhich was separated by chromatography. Finally, the product was workedup with K₂CO₃ in methanol to yield the (E)-alkene, depicted as compound2. The (E)-alkene, depicted as compound 2 of FIG. 2C, was then oxidizedin a multi-step process to yield the compound 3 (FIG. 2C)—an example ofthe (E)-alkene isostere.

The compound 3 of FIG. 2C was then conjugated with the peptideH-Pro-Val-Orn (Cbz)-OMe using 1-ethyl-3-(3-dimethylaminopropylcarbodimide hydrochloride) (EDC) as a coupling agent. The peptide is anexample of a suitable targeting sequence having affinity for themitochondria of a cell. The resulting product is shown as compound 4a inFIG. 2C. Saponification of compound 4a followed by coupling with4-amino-TEMPO (4-AT) afforded the resulting conjugate shown as compound5a in FIG. 2C in which the Leu-^(D)Phe peptide bond has been replacedwith an (E)-alkene.

In an alternate embodiment, conjugate 5b in FIG. 2C was prepared bysaponification and coupling of the peptide 4b(Boc-Leu-^(D)Phe-Pro-Val-Orn(Cbz)-OMe) with 4-AT. Similarly, conjugate5c in FIG. 2C was prepared by coupling the (E)-alkene isostere asindicated as compound 3 in FIG. 2C with 4-AT. These peptide and peptideanalogs are additional examples of suitable targeting sequences havingan affinity to the mitochondria of a cell.

In another embodiment, peptide isosteres may be employed as theconjugate. Among the suitable peptide isosteres are trisubstituted(E)-alkene peptide isosteres and cyclopropane peptide isosteres, as wellas all imine addition products of hydro- or carbometalated internal andterminal alkynes for the synthesis of di and trisubstituted (E)-alkeneand cyclopropane peptide isosteres. See Wipf et al., 2005b. Thesepeptide mimetics have been found to act as β-turn promoters. See Wipf etal., 2005b.

The linker, R2, may be any useful linker, chosen for its active groups,e.g., carboxyl, alkoxyl, amino, sulfhydryl, amide, etc. Typically, asidefrom the active groups, the remainder is non-reactive (such as saturatedalkyl or phenyl), and does not interfere, sterically or by any otherphysical or chemical attribute, such as polarity orhydrophobicity/hydrophilicity, in a negative (loss of function) capacitywith the activity of the overall compound. In one embodiment, aside fromthe active groups, the linker comprises a linear or branched saturatedC₄-C₂₀ alkyl. In one embodiment, the linker, R2 has the structure

In which n is 4-18, including all integers therebetween, in oneembodiment, 8-12, and in another embodiment, 10.

A person skilled in the organic synthesis arts can synthesize thesecompounds by crosslinking groups R1 and R3 by any of the manychemistries available. In one embodiment, R1 and R3 are to R2 by anamide linkage (peptide bond) formed by dehydration synthesis(condensation) of terminal carboxyl groups on the linker and an amine onR1 and R3 (or vice versa). In one embodiment, R1 and R3 are identical ordifferent and are selected from the group consisting of: XJB-5-131,XJB-5-125, XJB-7-75, XJB-2-70, XJB-2-300, XJB-5-208, XJB-5-197,XJB-5-194, .JP4-039 and JP4-049, attached in the manner shown in FIGS.2C and 2D.

Further examples of compounds which may be used according to thisinvention include, but are not limited to, compounds set forth in US20070161544 and US20070161573 (incorporated by reference in theirentireties herein), such as, for example, XJB-5-131.

5.2 Signs of Aging that May Be Modulated

The present invention may be used to inhibit the occurrence, progressionand/or severity of one or more signs of aging, including, but notlimited to, epidermal atrophy, epidermal hyperpigmentation, rhytid(wrinkles), photoaging of the skin, alopecia, hearing loss, visualimpairment, cerebral atrophy, cognitive deficits, trembling, ataxia,areflexia, cerebellar degeneration, hypertension, renal insufficiency,renal acidosis, incontinence, endocrinopathies, diabetes, decreasedliver function, hypoalbuminemia, hepatic accumulation of glycogen andtriglycerides, anemia, bone marrow degeneration, osteopenia,osteoporosis, kyphosis, degenerative joint disease, intervertebral discdegeneration, peripheral neuropathy, impaired wound healing increasedcellular senescence, retinal degeneration, motor neuron degeneration,cerebral lacunae, white matter degeneration, sarcopenia, muscleweakness, dystonia, increased peroxisome biogenesis, increasedapoptosis, decreased cellular proliferation, cachexia, and decreasedlifespan. “Inhibiting the occurrence, progression and/or severity of asign of aging” means reducing the risk of occurrence, delaying theonset, slowing the progression, and/or reducing the severity and/ormanifestation, of a sign of aging, and includes, but is not limited to,preventing the occurrence, development or progression of a sign ofaging.

The present invention may be used to improve age-related performance ina subject. “Improving performance” refers to any aspect of performance,including cognitive performance or physical performance, such as, butnot limited to, the ability to be self-sufficient, to take care of (somebut not necessarily all) personal needs, to be ambulatory or otherwisemobile, or interaction with others. In non-limiting embodiments, thepresent invention may be used to treat young individuals to preventage-related disease, and/or to treat young patients who have early onsetaging symptoms. A “young” individual, where the individual is a human,is less than about 60 years old, preferably between 10-59 years old, orbetween 20-50 years old. The present invention may also be used to treatpatients with syndromes of accelerated aging, e.g., progeria.

The present invention may be used to prolong the lifespan or healthspanof a geriatric subject, for example, relative to an age-matched,clinically comparable control not treated according to the invention.

5.3 Methods of Treatment

Accordingly, in one set of embodiments, the present invention providesfor a method of inhibiting one or more signs of aging in a subject inneed of such treatment, comprising administering, to the subject, aneffective amount of a compound as set forth above. “Inhibiting a sign ofaging” means reducing the risk of occurrence, delaying the onset,slowing the progression, and/or reducing the severity and/ormanifestation, of a sign of aging, and includes, but is not limited to,preventing the occurrence, development or progression of a sign ofaging.

In a related set of non-limiting embodiments, the present inventionprovides for a method of improving age-related performance in a subject,comprising administering to the subject an effective amount of acompound as set forth above.

In one non-limiting embodiment, the present invention provides for amethod of inhibiting a sign of aging in a young individual comprisingadministering to the subject an effective amount of a compound as setforth above.

In one non-limiting embodiment, the present invention provides for amethod of treating an early onset aging symptom in a young patientcomprising administering to the subject an effective amount of acompound as set forth above. In another non-limiting embodiment, thepresent invention provides for a method of treating an accelerated agingsyndrome in a subject in need of such treatment comprising administeringto the subject an effective amount of a compound as set forth above,wherein the accelerated aging syndromes include, but are not limited to,progerias. A “young” individual, where the individual is a human, isless than about 60 years old, preferably between 10-59 years old, orbetween 20-50 years old.

In another related set of embodiments, the present invention providesfor a method of prolonging survival of a geriatric subject, comprisingadministering, to the subject, an effective amount of a compound as setforth above.

In yet another set of embodiments, the present invention provides for amethod of inhibiting the occurrence, progression or severity of adegenerative disorder in a subject in need of such treatment, includingbut not limited to, a subject suffering from a neurodegenerativedisorder such as Alzheimer's Disease, Parkinson's Disease, AmyotrophicLateral Sclerosis, Huntington's Chorea, and Lewy Body Disease,osteoporosis, or a joint degenerative disorder, such as osteoarthriticor rheumatoid arthritis, or intervertebral disc degeneration comprisingadministering, to the subject, an effective amount of a compound as setforth above. The person skilled in the art would be aware of theappropriate indicia to measure to assess whether progression has beeninhibited for each of said degenerative disorders. Non-limiting specificexamples of indicia of neurodegeneration include impaired cognition,impaired short term memory, dystonia, trembling, choreoform movements,weakness, spasticity, decreased nerve conduction, ataxia, visualimpairment, peripheral neuropathy and hearing loss. “Inhibiting theoccurrence, progression or severity of a degenerative disorder” meansreducing the risk of occurrence, delaying the onset, slowing theprogression, and/or reducing the severity and/or manifestation, of adegenerative disorder, and includes, but is not limited to, preventingthe occurrence, development or progression of a degenerative disorder.

The compound may be administered systemically to achieve distributionthroughout the body or may be administered to achieve a local effect.The route of administration may be selected depending on the intendedeffect. As non-limiting examples, systemic administration, to achievetherapeutic levels throughout the body, may be achieved using aninhibitor suitable for distribution throughout the body, administeredvia any standard route, including but not limited to oral, intravenous,inhalation, intraperitoneal, subcutaneous, or intramuscular routes.Non-limiting examples of local administration include, but are notlimited to, intrathecal administration to treat central nervous systemmanifestations of aging, ocular instillation to treat visualdisturbances, intramuscular injection may be used to treat musclewasting, topical administration to prevent or reverse skin aging etc.

In one embodiment, the present invention provides for a method ofpreventing, delaying or attenuating loss of vision due to cataracts,leucoma, glaucoma or retinal degeneration in a subject in need of suchtreatment comprising administering, to the subject, an effective amountof a compound as set forth above.

In one embodiment, the present invention provides for a method oftreating accelerated aging syndromes in a subject in need of suchtreatment comprising administering, to the subject, an effective amountof a compound as set forth above, wherein the accelerated agingsyndromes include, but are not limited to progerias. In anotherembodiment, the present invention provides for a method of treatinggenome instability disorder in a subject in need of such treatmentcomprising administering, to the subject, an effective amount of acompound as set forth above, wherein the genome instability disordercauses accelerated aging of one or more tissues.

In yet another embodiment, the present invention provides for a methodof treating accelerated aging syndrome in a subject who has undergonecancer therapy comprising administering, to the subject, an effectiveamount of a compound as set forth above. In one embodiment, theaccelerated aging syndrome suffered by the subject is due to excessiveDNA damage caused by the cancer therapy undergone by the subject.Accelerated aging symptoms include but are not limited to, peripheralneuropathy, hair loss, greying, epidermal atrophy, poor wound healing,muscle wasting, loss of hearing, osteoporosis, trembling and cognitivedeficits.

In yet another embodiment, the present invention provides for a methodof preserving or improving quality and function of cell products whichare intended for therapy in a subject, which method comprisesadministering an effective amount of a compound as set forth above tothe cells prior to administering the cell products to the subject. Thetherapies include, but are not limited to, stem cell therapy, ips celltherapy, grafting, and autologous therapies.

Topical formulations may include administering the compound as set forthabove, optionally comprised in microsphere, microcapsule, or liposome,in a cream, lotion, organic solvent, or aqueous solution.

Compounds as set forth herein may be administered in a suitablepharmaceutical carrier (e.g. sterile water, normal saline, phosphatebuffered saline, etc.). Not by way of limitation, inhibitors may beadministered as a solution, as a suspension, in solid form, in asustained release formulation, in a topical cream formulation, etc. Inparticular non-limiting examples, an inhibitor may be incorporated intoa microcapsule, nanoparticle or liposome. For certain compounds,including, but not limited to, XJB-5-131, which are sparingly soluble,it may be desirable to use, as a carrier, an agent that promotespenetration through a cell membrane, such as, but not limited to,dimethylsulfoxide.

An effective dose/amount may be calculated by determining the amountneeded to be administered to produce a concentration sufficient toachieve the desired effect in the tissue to be treated, taking intoaccount, for example, route of administration, bioavailability,half-life, and the concentration which achieves the desired effect invitro or in an animal model system, using techniques known in the art.

Non-limiting examples of doses of compounds, as set forth herein,include between 0.1 and 50 mg/kg, or between 1 and 25 mg/kg, or between2 and 20 mg/kg, or about 2 mg/kg, or about 10 mg/kg, which may beadministered daily, at least 5 times a week, at least 3 times a week, atleast twice a week, at least once a week, at least twice a month, atleast once a month, at least once every three months, or at least onceevery six months.

In particular, non-limiting embodiments, a subject may be treated with acompound as set forth herein, using a regimen comprising a loadingperiod followed by a maintenance period, wherein the loading periodincludes treatment, with 1-20 mg/kg or 2-10 mg/kg, daily or every otherday for a period of 5-10 days, followed by a maintenance period whichincludes 1-10 mg/kg, or 10-50 mg/kg, given once a week, twice a week,three times a week, every other week, or once a month.

In other specific non-limiting embodiments where XJB-5-131 is thecompound, the dose may be between about 0.1 and 20 mg/kg, or betweenabout 0.3 and 10 mg/kg, or between about 2 and 8 mg/kg, or about 2mg/kg;

where either JP4-039, JED-E71-37 or JED-E71-58 is the compound, the dosemay be between about 0.01 and 50 mg/kg, or between about 0.1 and 20mg/kg, or between about 0.3 and 10 mg/kg, or between about 2 and 8mg/kg, or about 2 mg/kg;

a combination of any of the foregoing regimens may also be used; and

in any of the foregoing, the dose may be administered daily, at least 5times a week, at least 3 times a week, at least twice a week, at leastonce a week, at least twice a month, at least once a month, at leastonce every three months, or at least once every six months.

6. EXAMPLES 6.1. Example 1 A Murine Model of Aging

ERCC1-XPF is a DNA repair endonuclease that is essential for nucleotideexcision repair of bulky DNA adducts and the repair of DNA interstrandcrosslinks, and contributes to the repair of double-strand breaks(Sijbers et al., 1996; Niedemhofer et al., 2004; Ahmad et al., 2008).The two proteins are obligate binding partners required to stabilize oneanother in vivo (Niedernhofer et al., 2006) and are thought to functionexclusively as a nuclease in DNA repair (Sgouros et al., 1999).Mutations in Xpf that severely affect expression of ERCC1-XPF causedramatically accelerated aging in humans including the epidermal,hematopoietic, endocrine, hepatobiliary, nervous, musculoskeletal andcardiovascular systems (Niedernhofer et al., 2006). There are strongparallels between this progeroid syndrome and other diseases caused byinherited defects in genome maintenance mechanisms including Wernersyndrome, Cockayne syndrome, trichothiodystrophy, xeroderma pigmentosum,Rothmund Thompson syndrome and ataxia telangiectasia, all of whichinclude accelerated aging of one or more tissues (Hasty et al., 2003).This accelerated aging is generally accepted to be due to theaccumulation of unrepaired DNA damage (Garinis et al., 2008; Garinis etal., 2009).

As in humans, genetic depletion of Ercc1 or Xpf in the mouse causes asevere phenotype (McWhir et al., 1993; Tian et al., 2004; Weeda et al.,1997). Ercc1^(−/−) and Xpt^(−/−) mice die in the fourth week of lifewith symptoms associated with advanced age, including ataxia, kyphosis,weight loss, epidermal atrophy, sarcopenia, bone marrow degeneration,liver and kidney dysfunction, and evidence of replicative senescence(Niedemhofer et al., 2006; Weeda et al., 1997; Prasher et al., 2005).The comparison of Ercc1^(−/−) mice to naturally aged mice revealed ahighly significant correlation between the two at the level ofphysiology, histopathology and genome-wide expression patterns(Niedernhofer et al., 2006), which established these DNA repairdeficient mice as an accurate but accelerated model of natural aging,pertinent to humans because it mimics a human progeria. If the capacityto repair stochastic molecular damage is an important determinant oflifespan, then the prediction is that organisms with reduced capacityfor repair would have proportionally reduced lifespan. However, theextremely short lifespan of is these mice makes interventional studiesto probe the mechanism of aging impractical. To test this, a series ofmice were generated expressing various levels of ERCC1-XPF DNA repairendonuclease and therefore different capacities for DNA repair andlifespan. Hypomorphic and conditional ERCC1 mutants were cloned (FIG.3). The hypomorphic allele (A) contains a deletion of the last 7 aminoacids of ERCC1 to humanize the protein. Transcriptional 2 0 interferencefrom the neomycin cassette in the last intron of the gene reduces thestability of the Ercc1 mRNA by 6-fold (Weeda et al., 1997). In thesecond construct (cond), genomic sequence of Ercc1 exon 7 was fused to acDNA encoding exons 8-10, and the fusion was floxed with loxP sites toallow tissues specific deletion of ERCC1 in the mouse.

Ercc1^(−/Δ) compound heterozygote mice were bred and tissues isolatedfor analysis. Direct detection of ERCC1 protein is not currentlypossible due to the lack of an antibody that recognizes the murineprotein. As a surrogate, XPF, the obligate binding partner of ERCC1, wasmeasured in mouse liver (FIG. 4). XPF levels in Ercc1^(−/Δ) mice were10% that of wild type mice. In accordance, cells from the Ercc1^(−/Δ)had an intermediate sensitivity to DNA damage agents relative tocongenic wild type and ERCC1-null cells (FIG. 5). Furthermore,Ercc1^(−/Δ) mice had an intermediate lifespan relative to wild typelittermates and ERCC1 knock-out mice (FIG. 6A-D).

A cohort of Ercc1^(−/Δ) mice were allowed to live their full lifespanand monitored for symptoms associated with advanced age (FIG. 6B).Ercc1^(−/Δ) mice developed the same progeroid symptoms as Ercc1^(−/−)mice (and patients expressing low levels of ERCC1-XPF; FIG. 6C).However, the age at onset of symptoms was delayed from perinatal inErcc1−/− mice to 8 weeks of age in Ercc1^(−/Δ) mice (FIG. 6D). TheErcc1^(−/Δ) mice were healthy for the first 8-9 weeks of life, thenbegan to show numerous spontaneous and progressive symptoms associatedwith aging including signs of neurodegeneration, muscle wasting, loss ofvision and hearing, urinary incontinence, epidermal atrophy, bone marrowfailure, decreased liver and kidney function, loss of β islet cells,osteoporosis and intervertebral disc degeneration. FIG. 6D illustratestheir accelerated aging phenotype.

Genome-wide expression profiling in liver and pancreas of Ercc1^(−/Δ)mice established that the expression changes relative to wide typelittermate controls were nearly identical to, yet less dramatic thanthose of Ercc1^(−/−) mice (Schumacher et al., 2008, which in turncorrelated significantly to genome-wide expression changes seen withnatural aging (Niedernhofer et al., 2006). This emphasizes the relevanceof these models to natural aging.

6.2. Example 2 Protective Effects of XJB-5-131

To assess the effectiveness of XJB-5-131 in inhibiting degenerationand/or signs of aging and age-related degenerative diseases, thecompound was administered, over a 18-21 week period, to progeroidErcc1^(−/Δ) mice, at a dose of 2 mg/kg in sunflower seed oil carrier (topromote solubility) administered intraperitoneally three times per week(FIG. 7). Sunflower seed oil was administered to twin Ercc1^(−/Δ) miceaccording to the same schedule as a control. The treated and controlmice were monitored twice a week for weight and symptom/signdevelopment.

FIG. 8 presents a summary table showing the age at onset of progeroidsymptoms in Ercc1^(−/Δ) mice treated one example of the compounds asdisclosed herein, XJB-5-131 or vehicle only (oil). The aging scorereflected the relative age at onset of progeroid symptoms in one mousetreated with XJB-5-131 vs. its sibling treated with oil only and wasthus a measure of healthspan. In addition to improvement in most signsmeasured, the overall aging score was significantly improved in theXJB-5-131-treated mice. Treating Ercc1^(−/Δ) mice with XJB-5-131 delayedthe onset of progeroid symptoms and aging pathologies. Of note, all ofthe signs of neurodegeneration, including dystonia, trembling, ataxia,wasting and urinary incontinence, were delayed in the treated animalsproviding strong evidence that XJB-5-131 protects neurons againstdegenerative changes caused by oxidative stress.

FIG. 9A shows examples of twin ERCC1-deficient mice where one mouse wastreated with XJB-5-131 and its sibling received vehicle only. The mousetreated with XJB-5-131 showed reduced neurodegeneration and musclewasting as well as improved appearance.

To assess the ability of XJB-5-131 to inhibit deterioration ofintervertebral discs (an index of degenerative disease of thevertebrae), the level of glycosaminoglycan, an extra-cellular matrixprotein that is essential for disc maintenance and flexibility, in thediscs in treated and control mice were measured, and the results areshown in FIG. 9B. The intervertebral discs of treated mice containedapproximately 30 percent more glycosaminoglycan relative to controlmice, indicating delay of disc degeneration. In addition,immunohistochemical analysis of brains from mice demonstratedsignificantly reduced neurodegeneration in animals treated withXJB-5-131, compared to siblings treated with vehicle only (FIG. 9C).Also, XJB-5-131 treatment preserved insulin-producing B-islet cells inmice (FIG. 9D), cells necessary to prevent diabetes.

As a measure of the effect of XJB-5-131 on photoaging or sun-inducedskin changes, treated and control Ercc1^(−/cond). K 14-Cre mice, whichare missing ERCC1 only in the skin, were shaved, treated with adepilatory, then irradiated with UV-B light to induce sunburn (500 J/m2,the median erythemal dose). Subsequently, the mice were treated withXJB-5-131 (80 μg) emulsified in a cream daily for five days. Theresults, shown in FIG. 10A, indicated that the skin of XJB-5-131-treatedmice appeared much more smooth and healthy relative to cream-onlytreated control. In addition, immunofluorescence analysis of skinsections from these mice demonstrated that XJB-5-131 improved keratinand reduced inflammation of skin after acute or chronic photoaging(FIGS. 10B and C). In total, these data demonstrated the ability of acompound as disclosed herein to delay and ameliorate age-relateddegenerative changes of multiple organ systems.

At a macroscopic level, administration of XJB-5-131 appeared to havebeen well-tolerated by the animals, as indicated by the fact that theydid not lose weight as a result of treatment. Graphs showing weightversus time of treated, untreated and control animals are shown in FIG.11A-B. XJB-5-131 did not cause weight loss, as does the parental controlTEMPO. To assess the impact of XJB-5-131 at a cellular level, a numberof experiments were carried out using mouse embryonic fibroblasts(“MEF”) cells harvested from Ercc1^(−/−) mouse embryos. As shown in FIG.12, such MEF cultures were prepared and grown under ambient oxygen(oxidative stress) conditions, and then either untreated (media only) ortreated with a concentration of 500 nM (in media) XJB-5-131, and thentested for SA-β galactosidase staining (a marker of cellularsenescence). The amount of staining was notably less in the treatedcells. In addition, XJB-5-131 treatment was found to reduce the numberof γH2AX foci in DNA (a marker of double-stranded breaks; FIG. 13A),although it did not reduce the amount of apoptosis (FIG. 14).

6.3. Example 3 Protective Effects of JP4-039

To assess the therapeutic potential of one example of the compounds asdisclosed herein, JP4-039, tests for safety and protective activity wereperformed. FIGS. 15 and 16 show the results of tests to evaluate whethervarying concentrations of JP4-039 produce toxic effects after 48 hoursin cultures of MEF cells prepared from Ercc1^(−/−) or wild-type mouseembryos, respectively. Even under the highest concentrations tested (10μM), no signs of toxicity were observed in either culture system andcellular proliferation was enhanced relative to untreated control cells(media only).

To test the protective activity of JP4-039, cultures of primary MEF wereprepared from Ercc1^(−/−) mouse embryos and grown under 20% oxygen(ambient air), which creates oxidative stress in these cells that arehypersensitive to the reactive oxygen species. The cells were theneither treated with a concentration of 1 μM of XJB-5-131, JP4-039,JED-E71-37 or JED-E71-58, or left untreated, and then after 48 hours thelevel of p16, a marker of irreversible cellular senescence, was measuredby immunofluorescence staining. As seen in FIG. 17, the level of p16 wasmuch lower in MEF cells treated with JP4-039 relative to its level inuntreated cells, whereas XJB-5-131, JED-E71-37 and JED-E71-58 wereobserved to be less effective at this concentration. Furthermore, invivo, JP4-039 reversed epidermal atrophy, by increasing proliferation ofkeratinocytes (FIG. 10D). Thus JP4-039 was efficacious in vitro and invivo for reducing/reversing age-related degenerative changes.

6.4. Example 4 Protective Effects of the Compounds as Disclosed Hereinin Cell Culture

To evaluate the protective activities of two examples of the compoundsas disclosed herein, JED-E71-37 and JED-E71-58, primary MEF cells wereprepared from Ercc1^(−/−) mice and grown under conditions of oxidativestress (ambient air, 20% oxygen). The cells were then either untreatedor treated with 1 μM JED-E71-37 or JED-E71-58 for a period of 48 hours.As can be seen in FIG. 18, both compounds improved cell proliferationdespite the oxidative stress.

Next, the abilities of these two compounds, as well as XJB-5-131 andJP4-039, each at a concentration of 1 μM, were tested for their abilityto prevent cellular senescence in cell cultures prepared, andoxidatively stressed, as in the preceding paragraph. Treated as well asuntreated cells were, after 48 hours, immunostained for γ-H2AX, a markerof DNA double-strand breaks as well as cellular senescence. Results forJED-E71-58 are shown in FIG. 13, showing a distinct decrease in γ-H2AX.JP4-039 was also similarly effective, but XJB-5-131 and JED-E71-37 wereobserved to be less effective at this concentration. These datademonstrated the ability of these compounds as disclosed herein toattenuate two signs associated with aging (decreased cellularproliferation and increased cellular senescence).

6.5. Example 5 Protective Effects of XJB-5-131 in an Animal Model ofHuntington's Disease

To further assess whether XJB-5-131 has a protective effect againstneurodegeneration, the compound was tested in a second animal model.R6/2 mice model the neurodegenerative disease Huntington's disease.

Huntington's disease is a fatal autosomal dominant neurodegenerativedisease. The prevalence of Huntington's disease is four to seven in 100000 and it typically develops in mid-life. The symptoms involvepsychiatric, motor and cognitive disturbances, and weight loss.Huntington's disease is caused by an expansion of a CAG repeat in exon 1of the huntingtin gene, which encodes a protein suggested to beassociated with synaptic vesicles and microtubules in neurons. Mutanthuntingtin forms insoluble aggregates that accumulate in the cytoplasmand nucleus of cells, disrupting neuron function. R6/2 mice express exon1 of the human huntingtin gene, containing 150 CAG repeats.Consequently, the mice develop neurological symptoms that resemble manyof those seen in Huntington's disease, including deficits of motorco-ordination, altered locomotor activity, impaired cognitiveperformance and seizures. Symptoms begin by week 3-4 and areprogressive. A small cohort (n=5) of R6/2 mice were treated withXJB-5-131 according to the scheme in FIG. 7 (2 mg/kg, intraperitoneally,three times per week beginning at 5 weeks of age). Motor co-ordinationwas measured using a rotarod, after the mice were conditioned to theapparatus. R6/2 mice treated with XJB-5-131 had significantly bettermotor co-ordination at 6 and 7 weeks of age than untreated mutantanimals (FIG. 19). These data demonstrated that in a second animal modelof neurodegeneration, XJB-5-131 was efficacious in delaying theprogression of disease symptoms. Furthermore, the data indicated thatthe compounds as disclosed herein can specifically be used to treatHuntington's disease.

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Various publications are cited herein, the contents of which are herebyincorporated by reference in their entireties.

1. A method of inhibiting one or more signs of aging in a subject inneed of such treatment, comprising administering, to the subject, aneffective amount of a compound having a structure selected from thegroup consisting of Formula 1, Formula 2, and Formula 3, wherein thesign of aging is selected from the group consisting of the developmentor progression of one or more signs of aging, including, but not limitedto, epidermal atrophy, epidermal hyperpigmentation, wrinkles, hearingloss, visual impairment, cerebral atrophy, cognitive deficits,trembling, ataxia, cerebellar degeneration, hypertension, renalinsufficiency, renal acidosis, incontinence, decreased liver function,hypoalbuminemia, hepatic accumulation of glycogen and triglycerides,anemia, bone marrow degeneration, osteopenia, kyphosis, degenerativejoint disease, intervertebral disc degeneration, sarcopenia, muscleweakness, dystonia, increased peroxisome biogenesis, increasedapoptosis, decreased cellular proliferation, cachexia, and decreasedlifespan, wherein the Formula 1 has the following structure:

wherein X is

wherein R₁, R₂ and R₄ of Formula 1 are, independently, selected from agroup consisting of C₁-C₆ straight or branched-chain alkyl, optionallyincluding a phenyl (C₆H₅) group, that optionally is methyl-, hydroxyl-or fluoro-substituted, including including: methyl, ethyl, propyl,2-propyl, butyl, t-butyl, pentyl, hexyl, benzyl, hydroxybenzyl (e.g.,4-hydroxybenzyl), phenyl and hydroxyphenyl, wherein R₃ of Formula 1 is—NH—R₅, —O—R₅ or —CH₂—R₅, wherein R₅ is an —N—O. or —N—OH containinggroup, wherein R of Formula 1 is —C(O)—R₆ or —C(O)O—R₆, wherein R₆ isC₁-C₆ straight or branched-chain alkyl optionally comprising one or morephenyl (—C₆H₅) groups, and that optionally are methyl-, ethyl-,hydroxyl- or fluoro-substituted, including Boc (R═—C(O)O-tert-butyl) andCbz (R═—C(O)O-benzyl (Bn)) groups; wherein Formula 2 has the followingstructure:

wherein R1, R2 and R3 of Formula 2 are, independently, selected fromC₁-C₆ straight or branched-chain alkyl, optionally including aphenyl(C₆H₅) group which optionally is methyl-, hydroxyl- orfluoro-substituted, including 2-methyl propyl, benzyl, methyl-,hydroxyl- or fluoro-substituted benzyl, such as 4-hydroxybenzyl, whereinR4 of Formula 2 is an —N—O. or —N—OH containing group, wherein R ofFormula 2 is —C(O)—R₆ or —C(O)O—R5, wherein R5 is C₁-C₆ straight orbranched-chain alkyl, optionally comprising one or more phenyl (—C₆H₅)groups, and that optionally are methyl-, ethyl-, hydroxyl- orfluoro-substituted, including Boc and Cbz groups; wherein Formula 3 hasthe following structure

wherein R1, R2 and R3 of Formula 3 are, independently, selected fromC₁-C₆ straight or branched-chain alkyl, optionally including a phenyl(C₆H₅) group, that optionally is methyl-, hydroxyl- orfluoro-substituted, including 2-methyl propyl, benzyl, methyl-,hydroxyl- or fluoro-substituted benzyl, such as 4-hydroxybenzyl, whereinR4 of Formula 3 is an —N—O. or —N—OH containing group, wherein R ofFormula 3 is —C(O)—R5 or —C(O)O—R5, wherein R5 is C₁-C₆ straight orbranched-chain alkyl, optionally comprising one or more phenyl (—C₆H₅)groups, and that optionally are methyl-, ethyl-, hydroxyl- orfluoro-substituted, including Boc and Cbz groups.
 2. The method of claim1, wherein R₅ of Formula 1 is TEMPO (2,2,6,6-Tetramethyl-4-piperindine1-oxyl).
 3. The method of claim 1, wherein R₅ of Formula 1 is TEMPOL(4-Hydroxy-TEMPO).
 4. The method of claim 1, wherein R4 of Formula 2 isTEMPO (2,2,6,6-Tetramethyl-4-piperindine 1-oxyl).
 5. The method of claim4, wherein Formula 2 has one structure selected from the groupconsisting of


6. The method of claim 1, wherein R4 of Formula 3 is TEMPO(2,2,6,6-Tetramethyl-4-piperindine 1-oxyl).
 7. The method of claim 6,wherein Formula 3 has one structure selected from the group consistingof


8. The method of claim 1, wherein the compound is selected from thegroup consisting of XJB-5-131, JP4-039, JED-E71-37 and JED-E71-58.
 9. Amethod of inhibiting the progression of a degenerative disorder in asubject in need of such treatment, comprising administering, to thesubject, an effective amount of a compound having a structure selectedfrom the group consisting of Formula 1, Formula 2, and Formula 3,wherein the degenerative disorder is selected from the group consistingof a neurodegenerative disorder, a joint degenerative disorder, and abone degenerative disorder, wherein the Formula 1 has the followingstructure:

wherein X is

wherein R₁, R₂ and R₄ of Formula 1 are, independently, selected from agroup consisting of C₁-C₆ straight or branched-chain alkyl, optionallyincluding a phenyl (C₆H₅) group, that optionally is methyl-, hydroxyl-or fluoro-substituted, including including: methyl, ethyl, propyl,2-propyl, butyl, t-butyl, pentyl, hexyl, benzyl, hydroxybenzyl (e.g.,4-hydroxybenzyl), phenyl and hydroxyphenyl, wherein R₃ of Formula 1 is—NH—R₅, —O—R₅ or —CH₂—R₅, wherein R₅ is an —N—O. or —N—OH containinggroup, wherein R of Formula 1 is —C(O)—R₆ or —C(O)O—R₆, wherein R₆ isC₁-C₆ straight or branched-chain chain alkyl optionally comprising oneor more phenyl (—C₆H₅) groups, and that optionally are methyl-, ethyl-,hydroxyl- or fluoro-substituted, including Boc (R═—C(O)O-tert-butyl) andCbz (R═—C(O)O-benzyl (Bn)) groups; wherein Formula 2 has the followingstructure:

wherein R1, R2 and R3 of Formula 2 are, independently, selected fromC₁-C₆ straight or branched-chain alkyl, optionally including a phenyl(C₆H₅) group which optionally is methyl-, hydroxyl- orfluoro-substituted, including 2-methyl propyl, benzyl, methyl-,hydroxyl- or fluoro-substituted benzyl, such as 4-hydroxybenzyl, whereinR4 of Formula 2 is an —N—O. or —N—OH containing group, wherein R ofFormula 2 is —C(O)—R₆ or —C(O)O—R5, wherein R5 is C₁-C₆ straight orbranched-chain chain alkyl, optionally comprising one or more phenyl(—C₆H₅) groups, and that optionally are methyl-, ethyl-, hydroxyl- orfluoro-substituted, including Boc and Cbz groups; wherein Formula 3 hasthe following structure

wherein R1, R2 and R3 of Formula 3 are, independently, selected fromC₁-C₆ straight or branched-chain alkyl, optionally including a phenyl(C₆H₅) group, that optionally is methyl-, hydroxyl- orfluoro-substituted, including 2-methyl propyl, benzyl, methyl-,hydroxyl- or fluoro-substituted benzyl, such as 4-hydroxybenzyl, whereinR4 of Formula 3 is an —N—O. or —N—OH containing group, wherein R ofFormula 3 is —C(O)—R5 or —C(O)O—R5, wherein R5 is C₁-C₆ straight orbranched-chain chain alkyl, optionally comprising one or more phenyl(—C₆H₅) groups, and that optionally are methyl-, ethyl-, hydroxyl- orfluoro-substituted, including Boc and Cbz groups.
 10. The method ofclaim 9, wherein R₅ of Formula 1 is TEMPO(2,2,6,6-Tetramethyl-4-piperindine 1-oxyl).
 11. The method of claim 9,wherein R₅ of Formula 1 is TEMPOL (4-Hydroxy-TEMPO).
 12. The method ofclaim 9, wherein R4 of Formula 2 is TEMPO(2,2,6,6-Tetramethyl-4-piperindine 1-oxyl).
 13. The method of claim 12,wherein Formula 2 has one structure selected from the group consistingof


14. The method of claim 9, wherein R4 of Formula 3 is TEMPO(2,2,6,6-Tetramethyl-4-piperindine 1-oxyl).
 15. The method of claim 14,wherein Formula 3 has one structure selected from the group consistingof


16. The method of claim 9, wherein the degenerative disorder is selectedfrom the group consisting of Alzheimer's Disease, Parkinson's Disease,Amyotrophic Lateral Sclerosis, Lewy Body Disease, Huntington's Chorea,and Lewy Body Disease, osteoporosis, osteoarthritis and rheumatoidarthritis.
 17. The method of claim 9, wherein the compound is selectedfrom the group consisting of XJB-5-131, JP4-039, JED-E71-37 andJED-E71-58.
 18. A method of inhibiting loss of vision in a subject inneed of such treatment, comprising administering, to the subject, aneffective amount of a compound having a structure selected from thegroup consisting of Formula 1, Formula 2, and Formula 3, wherein theloss of vision is caused by one selected from the group consisting ofleucoma, glaucoma or retinal degeneration, wherein the Formula 1 has thefollowing structure:

wherein X is

wherein R₁, R₂ and R₄ of Formula 1 are, independently, selected from agroup consisting of C₁-C₆ straight or branched-chain alkyl, optionallyincluding a phenyl (C₆H₅) group, that optionally is methyl-, hydroxyl-or fluoro-substituted, including including: methyl, ethyl, propyl,2-propyl, butyl, t-butyl, pentyl, hexyl, benzyl, hydroxybenzyl (e.g.,4-hydroxybenzyl), phenyl and hydroxyphenyl, wherein R₃ of Formula 1 is—NH—R₅, —O—R₅ or —CH₂—R₅, wherein R₅ is an —N—O. or —N—OH containinggroup, wherein R of Formula 1 is —C(O)—R₆ or —C(O)O—R₆, wherein R₆ isC₁-C₆ straight or branched-chain chain alkyl optionally comprising oneor more phenyl (—C₆H₅) groups, and that optionally are methyl-, ethyl-,hydroxyl- or fluoro-substituted, including Boc (R═—C(O)O-tert-butyl) andCbz (R═—C(O)O-benzyl (Bn)) groups; wherein Formula 2 has the followingstructure:

wherein R1, R2 and R3 of Formula 2 are, independently, selected fromC₁-C₆ straight or branched-chain alkyl, optionally including a phenyl(C₆H₅) group which optionally is methyl-, hydroxyl- orfluoro-substituted, including 2-methyl propyl, benzyl, methyl-,hydroxyl- or fluoro-substituted benzyl, such as 4-hydroxybenzyl, whereinR4 of Formula 2 is an —N—O. or —N—OH containing group, wherein R ofFormula 2 is —C(O)—R₆ or —C(O)O—R5, wherein R5 is C₁-C₆ straight orbranched-chain chain alkyl, optionally comprising one or more phenyl(—C₆H₅) groups, and that optionally are methyl-, ethyl-, hydroxyl- orfluoro-substituted, including Boc and Cbz groups; wherein Formula 3 hasthe following structure

wherein R1, R2 and R3 of Formula 3 are, independently, selected fromC₁-C₆ straight or branched-chain alkyl, optionally including a phenyl(C₆H₅) group, that optionally is methyl-, hydroxyl- orfluoro-substituted, including 2-methyl propyl, benzyl, methyl-,hydroxyl- or fluoro-substituted benzyl, such as 4-hydroxybenzyl, whereinR4 of Formula 3 is an —N—O. or —N—OH containing group, wherein R ofFormula 3 is —C(O)—R5 or —C(O)O—R5, wherein R5 is C₁-C₆ straight orbranched-chain alkyl, optionally comprising one or more phenyl (—C₆H₅)groups, and that optionally are methyl-, ethyl-, hydroxyl- orfluoro-substituted, including Boc and Cbz groups.
 19. The method ofclaim 18, wherein R₅ of Formula 1 is TEMPO(2,2,6,6-Tetramethyl-4-piperindine 1-oxyl).
 20. The method of claim 18,wherein R₅ of Formula 1 is TEMPOL (4-Hydroxy-TEMPO).
 21. The method ofclaim 18, wherein R4 of Formula 2 is TEMPO(2,2,6,6-Tetramethyl-4-piperindine 1-oxyl).
 22. The method of claim 21,wherein Formula 2 has one structure selected from the group consistingof


23. The method of claim 18, wherein R4 of Formula 3 is TEMPO(2,2,6,6-Tetramethyl-4-piperindine 1-oxyl).
 24. The method of claim 23,wherein Formula 3 has one structure selected from the group consistingof


25. The method of claim 18, wherein the compound is selected from thegroup consisting of XJB-5-131, JP4-039, JED-E71-37 and JED-E71-58.
 26. Amethod of treating accelerated aging syndrome in a subject in need ofsuch treatment, comprising administering, to the subject, an effectiveamount of a compound having a structure selected from the groupconsisting of Formula 1, Formula 2, and Formula 3, wherein the Formula 1has the following structure:

wherein X is

wherein R₁, R₂ and R₄ of Formula 1 are, independently, selected from agroup consisting of C₁-C₆ straight or branched-chain alkyl, optionallyincluding a phenyl (C₆H₅) group, that optionally is methyl-, hydroxyl-or fluoro-substituted, including including: methyl, ethyl, propyl,2-propyl, butyl, t-butyl, pentyl, hexyl, benzyl, hydroxybenzyl (e.g.,4-hydroxybenzyl), phenyl and hydroxyphenyl, wherein R₃ of Formula 1 is—NH—R₅, —O—R₅ or —CH₂—R₅, wherein R₅ is an —N—O. or —N—OH containinggroup, wherein R of Formula 1 is —C(O)—R₆ or —C(O)O—R₆, wherein R₆ isC₁-C₆ straight or branched-chain alkyl optionally comprising one or morephenyl (—C₆H₅) groups, and that optionally are methyl-, ethyl-,hydroxyl- or fluoro-substituted, including Boc (R═—C(O)O-tert-butyl) andCbz (R═—C(O)O-benzyl (Bn)) groups; wherein Formula 2 has the followingstructure:

wherein R1, R2 and R3 of Formula 2 are, independently, selected fromC₁-C₆ straight or branched-chain alkyl, optionally including a phenyl(C₆H₅) group which optionally is methyl-, hydroxyl- orfluoro-substituted, including 2-methyl propyl, benzyl, methyl-,hydroxyl- or fluoro-substituted benzyl, such as 4-hydroxybenzyl, whereinR4 of Formula 2 is an —N—O. or —N—OH containing group, wherein R ofFormula 2 is —C(O)—R₆ or —C(O)O—R5, wherein R5 is C₁-C₆ straight orbranched-chain alkyl, optionally comprising one or more phenyl (—C₆H₅)groups, and that optionally are methyl-, ethyl-, hydroxyl- orfluoro-substituted, including Boc and Cbz groups; wherein Formula 3 hasthe following structure

wherein R1, R2 and R3 of Formula 3 are, independently, selected fromC₁-C₆ straight or branched-chain alkyl, optionally including a phenyl(C₆H₅) group, that optionally is methyl-, hydroxyl- orfluoro-substituted, including 2-methyl propyl, benzyl, methyl-,hydroxyl- or fluoro-substituted benzyl, such as 4-hydroxybenzyl, whereinR4 of Formula 3 is an —N—O. or —N—OH containing group, wherein R ofFormula 3 is —C(O)—R5 or —C(O)O—R5, wherein R5 is C₁-C₆ straight orbranched-chain alkyl, optionally comprising one or more phenyl (—C₆H₅)groups, and that optionally are methyl-, ethyl-, hydroxyl- orfluoro-substituted, including Boc and Cbz groups.
 27. The method ofclaim 26, wherein R₅ of Formula 1 is TEMPO(2,2,6,6-Tetramethyl-4-piperindine 1-oxyl).
 28. The method of claim 26,wherein R₅ of Formula 1 is TEMPOL (4-Hydroxy-TEMPO).
 29. The method ofclaim 26, wherein R4 of Formula 2 is TEMPO(2,2,6,6-Tetramethyl-4-piperindine 1-oxyl).
 30. The method of claim 29,wherein Formula 2 has one structure selected from the group consistingof


31. The method of claim 26, wherein R4 of Formula 3 is TEMPO(2,2,6,6-Tetramethyl-4-piperindine 1-oxyl).
 32. The method of claim 31,wherein Formula 3 has one structure selected from the group consistingof


33. The method of claim 26, wherein the accelerated aging syndrome isprogerias.
 34. The method of claim 26, wherein the subject has undergonecancer therapy.
 35. The method of claim 34, wherein the acceleratedaging syndrome suffered by the subject is due to excessive DNA damagecaused by the cancer therapy undergone by the subject.
 36. The method ofclaim 34 or 35 claim 34, wherein the accelerated aging syndrome isselected from the group consisting of peripheral neuropathy, hair loss,greying, epidermal atrophy, poor wound healing, muscle wasting, loss ofhearing, osteoporosis, trembling and cognitive deficits.
 37. The methodof claim 26, wherein the compound is selected from the group consistingof XJB-5-131, JP4-039, JED-E71-37 and JED-E71-58.
 38. A method oftreating genome instability disorder in a subject in need of suchtreatment, comprising administering, to the subject, an effective amountof a compound having a structure selected from the group consisting ofFormula 1, Formula 2, and Formula 3, wherein the genome instabilitydisorder is caused by accelerated aging syndrome, wherein the Formula 1has the following structure:

wherein X is

wherein R₁, R₂ and R₄ of Formula 1 are, independently, selected from agroup consisting of C₁-C₆ straight or branched-chain alkyl, optionallyincluding a phenyl (C₆H₅) group, that optionally is methyl-, hydroxyl-or fluoro-substituted, including including: methyl, ethyl, propyl,2-propyl, butyl, t-butyl, pentyl, hexyl, benzyl, hydroxybenzyl (e.g.,4-hydroxybenzyl), phenyl and hydroxyphenyl, wherein R₃ of Formula 1 is—NH—R₅, —O—R₅ or —CH₂—R₅, wherein R₅ is an —N—O. or —N—OH containinggroup, wherein R of Formula 1 is —C(O)—R₆ or —C(O)O—R₆, wherein R₆ isC₁-C₆ straight or branched-chain alkyl optionally comprising one or morephenyl (—C₆H₅) groups, and that optionally are methyl-, ethyl-,hydroxyl- or fluoro-substituted, including Boc (R═—C(O)O-tert-butyl) andCbz (R═—C(O)O-benzyl (Bn)) groups; wherein Formula 2 has the followingstructure:

wherein R1, R2 and R3 of Formula 2 are, independently, selected fromC₁-C₆ straight or branched-chain alkyl, optionally including a phenyl(C₆H₅) group which optionally is methyl-, hydroxyl- orfluoro-substituted, including 2-methyl propyl, benzyl, methyl-,hydroxyl- or fluoro-substituted benzyl, such as 4-hydroxybenzyl, whereinR4 of Formula 2 is an —N—O. or —N—OH containing group, wherein R ofFormula 2 is —C(O)—R₆ or —C(O)O—R5, wherein R5 is C₁-C₆ straight orbranched-chain alkyl, optionally comprising one or more phenyl (—C₆H₅)groups, and that optionally are methyl-, ethyl-, hydroxyl- orfluoro-substituted, including Boc and Cbz groups; wherein Formula 3 hasthe following structure

wherein R1, R2 and R3 of Formula 3 are, independently, selected fromC₁-C₆ straight or branched-chain alkyl, optionally including a phenyl(C₆H₅) group, that optionally is methyl-, hydroxyl- orfluoro-substituted, including 2-methyl propyl, benzyl, methyl-,hydroxyl- or fluoro-substituted benzyl, such as 4-hydroxybenzyl, whereinR4 of Formula 3 is an —N—O. or —N—OH containing group, wherein R ofFormula 3 is —C(O)—R5 or —C(O)O—R5, wherein R5 is C₁-C₆ straight orbranched-chain chain alkyl, optionally comprising one or more phenyl(—C₆H₅) groups, and that optionally are methyl-, ethyl-, hydroxyl- orfluoro-substituted, including Boc and Cbz groups.
 39. The method ofclaim 38, wherein R₅ of Formula 1 is TEMPO(2,2,6,6-Tetramethyl-4-piperindine 1-oxyl).
 40. The method of claim 38,wherein R₅ of Formula 1 is TEMPOL (4-Hydroxy-TEMPO).
 41. The method ofclaim 38, wherein R4 of Formula 2 is TEMPO(2,2,6,6-Tetramethyl-4-piperindine 1-oxyl).
 42. The method of claim 41,wherein Formula 2 has one structure selected from the group consistingof


43. The method of claim 38, wherein R4 of Formula 3 is TEMPO(2,2,6,6-Tetramethyl-4-piperindine 1-oxyl).
 44. The method of claim 43,wherein Formula 3 has one structure selected from the group consistingof


45. The method of claim 38, wherein the accelerated aging syndrome isprogerias.
 46. The method of claim 38, wherein the compound is selectedfrom the group consisting of XJB-5-131, JP4-039, JED-E71-37 andJED-E71-58.